γ-Secretase is a complex protein (aspartate protease) comprising presenilin, nicastrin, Aph-1 and Pen-2 as basic components. Presenilin is the catalytic domain; presenilin gene has been identified as a causative gene for familial Alzheimer's disease (AD). γ-Secretase acts on single-pass transmembrane proteins as its substrates. As most representative substrates thereof, amyloid precursor protein (APP) and Notch are known. When cleaved by β-secretase at β-site and by γ-secretase at γ-site, APP produces amyloid β protein (Aβ). The thus-produced Aβ is classified into peptides with different lengths depending on the cleavage site in the amino acid sequence (C-terminal side). Of these peptides, Aβ42 which is strongly hydrophobic and ready to aggregate (ready to take the β-sheet structure) exhibits neurotoxicity. It has been considered that this phenomenon may be the major cause of Alzheimer's disease. Recently, however, a report has been made that presenilin 1 (PS1) and presenilin 2 (PS2) double-knockout mice capable of producing no Aβ show AD-like phenotypes such as decrease in synapses and neuronal death; this suggests existence of a pathogenic mechanism of AD independent from APP (Non Patent Document 1).
On the other hand, Eph receptor A4 (EphA4) is a member of the receptor tyrosine kinase family, and is a molecule regulating the morphogenesis of postsynapses. It is known that EphA4 knockout or expression of EphA4 dominant-negative mutants causes decrease in the number of spines (small, thorn-like protrusions found on dendrites) and makes the spine shape slender (Non Patent Document 2). It is generally proposed that the processes of memory and learning are reflected on the number and morphology of spines.
As molecules known to be involved in neural activity, Rho family proteins which are small GTP-binding proteins belonging to the Ras super family are known to regulate rearrangement of intracellular skeletons, particularly actin cytoskeleton. As representative molecules of Rho family proteins, RhoA, Cdc42 and Rac1 may be enumerated. It is known that these molecules are strongly expressed in the central nervous system. Especially, in spines where rearrangement of actin is vigorously performed, RhoA, Cdc42 and Rac1 are playing central roles. It is known that when these signal transduction pathways are broken, spines are attenuated and various neuronal disorders are caused (Non Patent Document 3). In particular, Rac-mediated signal transduction pathway functions to promote spine formation and, when its signals are inhibited, attenuation of spines occurs (Non Patent Document 5). It has already been reported that EphA4 is involved in regulation of the activities of Rho family molecules. When EphA4 is bound to ephrinA (ligand of EphA4 receptor), i.e., in a state that EphA4 is not processed by γ-secretase, EphA4 is known to inhibit Rac-mediated signal transduction pathway to thereby inhibit spine formation (Non Patent Document 4). Further, it is also known that Rac is deeply involved in the pathogenic mechanism of Alzheimer's disease. It is reported that p21-activated kinase (PAK) whose activity is regulated by Rac is decreased in both expression and activity in the brain of Alzheimer patients (Non Patent Document 6).
However, no report has been made to date as to the relationship between EphA4 and γ-secretase. It has not been reported that γ-secretase processes EphA4. Furthermore, no report has been made also as to the identification of EphA4 fragments processed by γ-secretase and the effect of such fragments in the body, especially the effect of EphA4 processing by γ-secretase upon the activities of Rho family molecules and upon spine formation.
[Non Patent Document 1] Saura C A, Choi S Y, Beglopoulos V, Malkani S, Zhang D, Shankaranarayana Rao B S, Chattarji S, Kelleher R J 3rd, Kandel E R, Duff K, Kirkwood A, and Shen J., Loss of presenilin function causes impairments of memory and synaptic plasticity followed by age-dependent neurodegeneration. Neuron. 2004 Apr. 8; 42(1):23-36.[Non Patent Document 2] Murai K K, Nguyen L N, Irie F, Yamaguchi Y, Pasquale E B. Control of hippocampal dendritic spine morphology through ephrin-A3/EphA4 signaling. Nat. Neurosci. 2003 February; 6(2):153-60.[Non Patent Document 3] Ramakers G J. Rho proteins, mental retardation and the cellular basis of cognition. Trends Neurosci. 2002 April; 25(4):191-9.[Non Patent Document 4] Shamah S M, Lin M Z, Goldberg J L, Estrach S, Sahin M, Hu L, Bazalakova M, Neve R L, Corfas G, Debant A, Greenberg M E., EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin. Cell. 2001 Apr. 20; 105(2):233-44.[Non Patent Document 5] Tashiro A, Yuste R., Regulation of dendritic spine motility and stability by Rac1 and Rho kinase: evidence for two forms of spine motility. Mol Cell Neurosci. 2004 July; 26(3):429-40.[Non Patent Document 6] Zhao L, Ma Q L, Calon F, Harris-White M E, Yang F, Lim G P, Morihara T, Ubeda O J, Ambegaokar S, Hansen J E, Weisbart R H, Teter B, Frautschy S A, Cole G M. Role of p21-activated kinase pathway defects in the cognitive deficits of Alzheimer disease. Nat. Neurosci. 2006 February; 9(2):234-42. Epub 2006 Jan. 15.